Actin Filaments

9 intracellular locations of actin filaments 

1. Microvilli

2. Cell cortex

3. Adherens belt

4. Filopodia

5. Lamellipodium

6. Stress fibres

7. Phagocytosis

8. Moving endocytic vesicles 

9. Contractile ring

How do actin filaments facilitate cell movement?

Actin filaments extend the lamellipodium and pull the cell forward
The leading edge pushes forward and stress fibres pull up the rear 

1. Extension

   1. Polymerize actin and protrude

   2. Focal adhesion 

2. Adhesion 

   1. Extend lamellipodium

3. Translocation 

   1. Pull cell body forward

4. De-adhesion and endocytic recycling

Phases of actin polymerization

0. Actin is diffusing around in solution: G-actin (globular actin is a protein by itself)

1. Nucleation: 3 actin subunits collide in such a way that they collide and form a nucleus for the polymerization of actin (Slow)

2. Elongation: Protein polymerizes 

   1. Actin subunits collide with the nucleus

   2. F-actin: filamentous actin

3. Steady state: New actin keeps colliding

(+) end of actin filament's on rate constant

12 µM^-1s^-1

(+) end of actin filament's off rate constant

1.4 s^-1

(+) end of actin filament's critical constant

C⁺c = 0.12 µM

(-) end of actin filament's on rate constant

1.3 µM^-1s^-1

(-) end of actin filament's off rate constant

0.8 s^-1

(-) end of actin filament's critical constant

C^-c = 0.60 µM

What happens to the concentration of free actin as a polymer grows?

The concentration of free actin decreases

What happens when the concentration of free actin decreases to C^-c?

The (-) end reaches equilibrium

Why does the (+) end of the polymer continue to grow even after the (-) end reaches equilibrium?

Because the concentration of free actin is still above C⁺c for the (+) end

What happens when the concentration of free actin drops below the C^-c of the (-) end?

The (-) end starts shrinking to maintain equilibrium

Actin is an ______ that drives cell motility 

Actin is an ATPase that drives cell motility 

How do actin filaments contribute to the lamellipodium?

Directly behind filopodia

How do actin filaments contribute to stress fibres?

At the back end of the cell

What features of actin filaments can cells control?

LANBO

• Length

• Angle

• Number

• Bundling

• Orientation

Structure of microfilaments

• Built by actin

• Two stranded polymer

• Forms thick bundles: stress fibres

• 7-9 nm thick

Actin's 2-stranded helical filament structure 

• 36 nm for one helical pitch 

• Polarized filaments

  • (+): smooth end

  • (-): notched end

How do actin filaments contribute to the adherens belt?

Seals from fluids leaking

How do actin filaments contribute to the contractile ring?

When cells are undergoing cytokinesis, they allow for the cell to cleave into two cells

How do actin filaments contribute to the cell cortex?

They line the outer edge of the cell

How do actin filaments contribute to the filopodia?

At the leading edge, driving motion

How do actin filaments contribute to phagocytosis?

When the cell is engulfing, they outline the site of entry

Why is the off rate constant lower than the on rate constant on the (+) end?

Probability of fall off is driven not by concentration but by random kinetic events like ion and water colliding with the filament, which is less likely

What is the critical constant?

C⁺c is the concentration at which on and off events are equal

What is the process called when the (+) end grows while the (-) end shrinks in a polymer?

Treadmilling

In actin treadmilling, the _____ stays the same, the _____ grows and the ____ shrinks.

In actin treadmilling, the length stays the same, the (+) end grows and the (-) end shrinks.

Formula to calculate net growth

Net growth = subunits (+) - subunits (-)

Net growth = k_ON [subunits] - k_OFF

• (+): on rate const. * [subunits]

• (-): off rate const.

Formula to calculate Cc

Cc = k_OFF / k_ON

• Net growth = 0

5 Main actin regulators

1. Formins

2. Cofilin

3. Capping protein

4. Arp 2/3

5. ⍺ - actinin

Formins

Grow the filaments by accelerating the rate of actin growth

Cofilin

Cut the filament / fragment it

Capping proteins

Stop it from growing and polymerizing

Arp 2/3

Make branched network

⍺ - actinin

Cross-link the filaments

Function of cross-linkers at binding domains

Regulate the spacing and orientation of resulting bundle

Cross-linkers at binding domains

1. Fimbrin

2. ⍺ - actinin

3. Spectrin

4. Filamin

Fimbrin

• Local adhesion

• Location: microvilli, filopodia, focal adhesions

⍺ - actinin

• Dimer

• Each polypeptide has 1 actin binding domain

• Location: stress fibers, filopodia, muscle Z line

Spectrin

• Spectral actin network

• Heterotrimer, dimer

• Location: Cell cortex

Filamin

• Singular polypeptide

• Flexible kink

• Location: Leading edge, stress fibres, filopodia

How does actin allow for pathogenic bacteria to move?

• Actin makes a comet tail for the bacteria to move around to break out of the cell and infect other cells

• Listeria “comet tails” are nucleated by the Arp 2/3 complex

How does Arp 2/3 function?

• Mimics an actin nucleus (when polymerizing)

• Dimer comes together when activated on the daughter filament

• Attaches at 70º

  • Brownian ratchet: Presses up against the PM until it gets stuck

What activates the Arp 2/3 complex?

WASp delivers the actin monomer to Arp 2/3

What are the domains of WASp?

1. WH2 domain

2. A domain

3. C domain

What does the WH2 domain in WASp do?

Binds to the actin monomer

What does the A domain in WASp do?

Acidic domain binding the Arp 2/3 complex

• Conformational change bringing it together

What main challenges does Lamellipodia run into during activity?

1. Running out of actin

2. Preventing futile polymerization

How does the cell make sure that it doesn’t run out of actin?

It keeps the concentration of actin relative to the critical constant

How does the cell make sure that there is no futile polymerization?

• Don’t grow actin far away

• Don’t want actin polymerizing at the (-) end

What protein aids the complications of Lamellipodia? How does it function?

Prolifin

• Recycles actin

• Maintain high concentration

• Blocks (-) end polymerization

• ADP-G actin refreshed and accelerates rate of ATP exchange

What protein severs actin filaments?

ADF/Cofilin

• At the (-) end

• Unwinds the actin and creates instability

Together, what proteins accelerate actin filament treadmilling?

• Cofilin: depolarizes

• Profilin: recharges

• Thymosin - β4: sequesters actin in reverse

What proteins prevent futile polymerization? How?

Capping protein

• Caps the actin filament so that new actin doesnt bind

• (+) end: Cap Z

• (-) end: Tropomodulin

How do formins polymerize linear actin filaments?

• Dimer sits on top of actin

  • Moves up as actins are added to the filament

  • FH2: Domains sit at the end

  • FH1: Long slender arms that bind to profilin actin — feeding

• Adds >10,000 monomers before detaching

What is myosin?

Myosin is a motor protein that produces force by converting chemical energy into mechanical energy

What experiment demonstrates myosin’s ability to translocate actin filaments?

In experiments where myosin heads attach to a glass slide, actin filaments move, with the (-) end moving forward as myosin heads try to move towards the (+) end

What are the three classes of myosin? Step sizes?

• Class I: 10-14 nm

• Class II: 5-10 nm

• Class V: 36 nm

What is the primary function of Class I myosin?

Class I myosin is involved in membrane association with the actin cytoskeleton and endocytosis

What is the primary function of Class II myosin?

Class II myosin is responsible for contraction

What is the primary function of Class V myosin?

Class V myosin binds to organelles like vesicles and facilitates their transport

What does non-muscle myosin-II do to actin filaments?

Non-muscle myosin-II forms bundles that pull actin filaments inward. Filaments at the ends create inward directional force that pushes actin inward.

How do myosin-II bundles contribute to cellular movement?

Myosin-II bundles contract actin arrays, enabling cytoskeletal rearrangement necessary for movement.

What is Dictyostelium, and why is it significance?

Dictyostelium is an amoeba that aggregates with other amoebae during starvation, demonstrating cellular communication and collective behavior.

How does Dictyostelium respond to starvation?

Sends stress signals that prompt other amoebae to produce fruiting bodies, allowing some members to relocate to areas with more food.

What chemical signal does Dictyostelium use for movement, and how does it work?

Dictyostelium uses cyclic AMP (cAMP). Cells send out cAMP signals, and other amoebae move toward the higher concentration of cAMP

Why do myosin heads move toward the (+) end of actin filaments?

Myosin heads generate mechanical force by hydrolyzing ATP, propelling them toward the (+) end for filament sliding

What happens to actin filaments when myosin-II bundles contract?

Contraction of myosin-II bundles pulls actin filaments inward, creating mechanical force for cellular functions like cytokinesis

What is the relationship between myosin-II and cellular contraction?

Myosin-II’s ability to form bundles and pull actin filaments inward enables the contraction of actin arrays, a key process in cellular movement and division.

What are the 3 types of Rho family GTPase? In short, what do each do?

1. Dominant active Rho: actin —> stress fibers

2. Dominant active Rac: Causes cells to grow lamellopodia

3. Dominant active Cdc42: produce filopodia

How do Rho GTPase translate outside signals into changes in the actin cytoskeleton?

1. A receptos binds to an extracellular signal (e.g. cAMP)

2. The Rho fam. GTPase is initially in an inactive GDP-bound state

3. The receptor interacts with a GEF
   (GDP —> GTP on Rho) to go into active state

4. Rho undergoes a conformational change: interact with effector proteins

5. Effector proteins (e.g. formins and Arp 2/3) regulate the actin cytoskeleton

6. To ensure precise control, cells need to toggle the activity of Rho proteins on and off

7. To deactivate Rho, GAP triggers the hydrolysis of GTP —> GDP

How does Rho activate formins?

Relieves formin auto-inhibition. RBD blocks FH2 when folded, but the binding of Rho unfolds.

How does Cdc42 activate Arp 2/3?

Opens WASp, activate Cdc42 interacts with WASp and RBP, activating Arp 2/3

How do extracellular signals in cells create zones of different Rho activity?

• Cdc42 activation at front

• Front/Leading edge: Rac activation leads to Arp 2/3 activation

  • Actin filaments assemble to treadmilling

• Back: Rho activation leads to Myosin II activation

  • Contraction of myosin II filaments in both stress fibres and cell cortex

Chemotactic gradient

Concentration of cAMP is higher at the leading edge

What mechanism in the cell allows for the back to become “Rho exclusive”?

• Wave of Rac propagates back —> Rho —> stress fibres

• Rho pushes back on Rac —> back = Rho exclusive